Abstract Acute allograft rejection is a T cell mediated immune response that continues to undermine the success of transplantation to treat end-stage organ disease. Two factors contributing to acute and chronic allograft injury and graft loss are ischemia-reperfusion injury and heterologous immunity by endogenous memory T cells with reactivity to graft alloantigens. Reperfusion of ischemic tissues is an inherent component of transplantation and quickly induces an acute inflammatory response that directs the infiltration and activation of many types of leukocytes in the tissue. Clinically, the presence of donor-reactive memory T cells in the peripheral blood of kidney transplant patients prior to transplant is associated with increased delayed graft function and poorer graft survival. However, the mechanisms activating donor-reactive endogenous memory T cells within allografts to mediate graft injury remain poorly understood. Studies performed during the last funding period indicated that endogenous memory CD8 T cells rapidly infiltrate cardiac allografts and are activated by graft allogeneic class I MHC molecules to proliferate and produce IFN-? and other factors mediating graft tissue injury. The infiltration and activities of endogenous memory CD8 T cells are markedly increased in allografts subjected to longer duration of cold ischemic storage (CIS) prior to transplant that promotes costimulatory blockade resistant memory CD8 T cell rejection of the allografts. These and our preliminary results have led us to propose the hypothesis that the increased inflammation generated following reperfusion of allografts subjected to prolonged, but not to minimal, CIS initiates a cycle of interdependent innate immune interactions with pre-existing donor-reactive memory CD4 and CD8 T cells required to sustain activation of the memory CD8 T cells to mediate acute graft injury and CTLA-4Ig-resistant rejection. The hypothesis will be tested in two specific aims: first, we will test the mechanisms promoting activation of the donor-reactive endogenous memory CD8 T cells within the highly ischemic allografts; and, second, we will test the role of TLR9 signaling in sustaining the inflammatory cycle leading to activation of the memory CD8 T cells to reject the allografts and the ability of new strategies to inhibit TLR9-mediated inflammation to improve outcomes of these higher risk allografts. Experiments in this renewal application will connect ischemia-reperfusion injury with pre-existing donor-reactive memory T cell activation in grafts and provide novel insights into mechanisms underlying an important and poorly understood clinical problem. We anticipate these studies will identify new targets for therapeutic strategies to inhibit innate immune enhancement of pre-existing donor-reactive memory T cells in grafts and acute T cell mediated graft injury and improve graft outcomes.